Flavonoids have shown anticarcinogenic activity in cancer cell lines, animal models, and some human studies. Quantitative structure-activity relationship (QSAR) models have become useful tools for identification of promising lead compounds in anticancer drug development. However, epidemiological and clinical studies are still scarce. Compounds with flavonoid scaffold have been the subject of many mechanistic studies in cells, but information on human chemopreventive properties is still missing. The knowledge of the mechanisms of action, anti-multidrug resistance, and QSAR studies on flavonoids and related compounds may help to enhance research on these compounds and their bioactivity. Therefore, once the issue is introduced, the mechanisms involved, and QSAR studies developed to predict the activity and toxicity of these chemicals to biological systems are discussed. QSAR studies on flavonoids as inhibitors of breast cancer resistance protein (BCRP/ABCG2), 17β-hydroxysteroid dehydrogenase (17β-HSD), PIM-1 kinase and cyclin-dependent kinases (CDKs) are analyzed. Combined treatment of flavonoids with TRAIL and current chemotherapy agents is also discussed as a promising cancer chemoprevention and/or therapy.

Chronic myeloid leukemia (CML) is a myeloproliferative syndrome characterizedby the presence of the Philadelphia chromosome which encodes a constitutivelyactivated tyrosine kinase (BCRABL).The first line treatment for CML consistson BCRABLinhibitors such as Imatinib. Nevertheless, such treatment may leadto the selection of resistant cells. Therefore, it is of great value to find moleculesthat enhance the antiproliferativeeffect of firstlinedrugs. Hyaluronan is themain glycosaminglican of the extracellular matrix which is involved in tumorprogression and multidrug resistance. We have previously demonstrated thatthe inhibition of hyaluronan synthesis by 4methylumbelliferone(4MU) inducessenescence and can revert Vincristine resistance in CML cell lines. However, theeffect of 4MU on Imatinib therapy remains unknown. The aim of this work was todetermine whether the combination of 4MU with Imatinib is able to modulatethe proliferation as well as apoptosis and senescence induction in human CMLcell lines. For this purpose the ATCC cell line K562, and its multidrug resistantderivate, Kv562 were used. Cells were exposed to 4MU, Imatinib or acombination of both. We demonstrated that 4MU and Imatinib cotreatmentabrogated the proliferation of both cell lines. However, such cotreatmentdid notincrease the levels of apoptosis when compared with the treatment with Imatinibalone. For both cell lines the mechanisms of tumor suppression involved wassenescence, since the combination of 4MU and Imatinib arrested the cell cycle and increased senescence associated βgalactosidaseactivity and senescenceassociated heterochromatin foci presence when compared to each drug alone.Moreover, 4MU, Imatinib and 4MU + Imatinib decreased pAkt/Akt ratio in bothcell lines and reduced the pERK/ERK ratio only in K562 cells. These findingshighlight the potential use of 4MU together with Imatinib for CML therapy.

The objectives of this study were: i) to control the resistance of E. coli against various commonly used antimicrobials for therapeutic and prophylactic in pig farms ii) to isolate and characterize phenotypic and genotypic E. coli toxigenic diarrhea from pigs pre and post weaning, iii) to determinate the presence of class 1 and 2 integrons as possible resistance mechanism of spread of E. coli from porcine. Rectal swabs were processed from 216 clinically healthy and diarrheic pigs, from 15 pig farms. 46.6% of the isolates were resistant to multiple antimicrobials, 93% were resistant to tetracycline, 59% to ciprofloxacin, 52% to florfenicol, 8% to amoxicillin/clavulanic acid and 0.6% to gentamicin. No resistance to colistina was observed. Out of 56 E. coli, 34 carried at least one gene int2 or int1. Toxigenic E. coli was isolated from 53% of pigs with diarrhea. The inappropriate use of antimicrobial to prophylactic or therapeutic purposes in veterinary medicine, involves a risk to public health.

Estradiol-17β-d-glucuronide (E17G), through the activation of different signaling proteins, induces acute endocytic internalization of canalicular transporters in rat, including multidrug resistance-associated protein 2 (Abcc2) and bile salt export pump (Abcb11), generating cholestasis. Insulin-like growth factor 1 receptor (IGF-1R) is a membrane-bound tyrosine kinase receptor that can potentially interact with proteins activated by E17G. The aim of this study was to analyze the potential role of IGF-1R in the effects of E17G in isolated perfused rat liver (IPRL) and isolated rat hepatocyte couplets. In vitro, IGF-1R inhibition by tyrphostin AG1024 (TYR, 100 nM), or its knock-down with siRNA, strongly prevented E17G-induced impairment of Abcc2 and Abcb11 function and localization. The protection by TYR was not additive to that produced by wortmannin (PI3K inhibitor, 100 nM), and both protections share the same dependency on microtubule integrity, suggesting that IGF-1R shared the signaling pathway of PI3K/Akt. Further analysis of the activation of Akt and IGF-1R induced by E17G indicated a sequence of activation GPR30-IGF-1R-PI3K/Akt. In IPRL, an intraportal injection of E17G triggered endocytosis of Abcc2 and Abcb11, and this was accompanied by a sustained decrease in the bile flow and the biliary excretion of Abcc2 and Abcb11 substrates. TYR did not prevent the initial decay, but it greatly accelerated the recovery to normality of these parameters and the reinsertion of transporters into the canalicular membrane. In conclusion, the activation of IGF-1R is a key factor in the alteration of canalicular transporter function and localization induced by E17G, and its activation follows that of GPR30 and precedes that of PI3K/Akt.

Multidrug resistance-associated protein 2 (MRP2) is an ATP-dependent transporter expressed at the brush border membrane of the enterocyte that confers protection against absorption of toxicants from foods or bile. Acute, short-term regulation of intestinal MRP2 activity involving changes in its apical membrane localization was poorly explored. We evaluated the effects of dibutyryl-cAMP (db-cAMP), a permeable analog of cAMP, and estradiol-17β-d-glucuronide (E217G), an endogenous derivative of estradiol, on MRP2 localization and activity using isolated rat intestinal sacs and Caco-2 cells, a model of human intestinal epithelium. Changes in MRP2 localization were studied by Western blotting of plasma membrane (PM) vs. intracellular membrane (IM) fractions in both experimental models, and additionally, by confocal microscopy in Caco-2 cells. After 30 min of exposure, db-cAMP-stimulated sorting of MRP2 from IM to PM both in rat jejunum and Caco-2 cells at 10 and 100 µM concentrations, respectively, with increased excretion of the model substrate 2,4-dinitrophenyl-S-glutathione. In contrast, E217G (400 µM) induced internalization of MRP2 together with impairment of transport activity. Confocal microscopy analysis performed in Caco-2 cells confirmed Western blot results. In the particular case of E217G, MRP2 exhibited an unusual pattern of staining compatible with endocytic vesiculation. Use of selective inhibitors demonstrated the participation of cAMP-dependent protein kinase and classic calcium-dependent protein kinase C in db-cAMP and E217G effects, respectively. We conclude that localization of MRP2 in intestine may be subjected to a dynamic equilibrium between plasma membrane and intracellular domains, thus allowing for rapid regulation of MRP2 function.

Accumulation and toxicity of cyanobacterial toxins, particularly microcystin-LR (MCLR) have been extensively studied in fish and aquatic invertebrates. However, MCLR excretion mechanisms, which could reduce this toxin's effects, have received little attention. The Patagonian silverside, Odontesthes hatcheri, is an omnivorous-planktivorous edible fish, which has been shown to digest cyanobacterial cells absorbing MCLR and eliminating the toxin within 48 h without suffering significant toxic effects. We studied the effects of MCLR on glycoconjugate composition and the possible role of multidrug resistance associated proteins (Abcc) in MCLR export from the cells in O. hatcheri intestine. We treated O. hatcheri with 5 μg MCLR g−1 body mass administered with the food. Twenty four hours later, the intestines of treated and control fish were processed for lectin-histochemistry using concanavalin A (ConA), Triticum vulgaris agglutinin (WGA), and Dolichos biflorus agglutinin (DBA). MCLR affected the distribution of glycoconjugates by augmenting the proportion of ConA-positive at the expense of WGA-positive cells. We studied MCLR effects on the transport of the Abcc-like substrates 2,4-dinitrophenyl-S-glutathione (DNP-SG) and calcein in ex vivo intestine preparations (everted and no-everted sacs and strips). In treated preparations, CDNB together with MCLR (113 μg MCLR g−1 intestine, equivalent to 1.14 μmol L−1 when applied in the bath) or the Abcc inhibitor, MK571 was applied for one hour, during which DNP-SG was measured in the bath every 10 min in order to calculate mass-specific DNP-SG transport rate. MCLR significantly inhibited DNP-SG transport (p < 0.05), especially in middle intestine (47 and 24%, for luminal and serosal transport, respectively). In middle intestine strips, MCLR and MK571inhibited DNP-SG transport in a concentration dependent fashion (IC50 3.3 and 0.6 μmol L−1, respectively). In middle intestine strips incubated with calcein-AM (0.25 μmol L−1), calcein efflux was inhibited by MCLR (2.3 μmol L−1) and MK571 (3 μmol L−1) by 38 and 27%, respectively (p < 0.05). Finally, middle intestine segments were incubated with different concentrations of MCLR applied alone or together with 3 μM MK571. After one hour, protein phosphatase 1 (PP1) activity, the main target of MCLR, was measured. 2.5 μM MCLR did not produce any significant effect, while the same amount plus MK571 inhibited PP1 activity (p < 0.05). This effect was similar to that of 5 μM MCLR. Our results suggest that in O. hatcheri enterocytes MCLR is conjugated with GSH via GST and then exported to the intestinal lumen through Abcc-like transporters. This mechanism would protect the cell from MCLR toxicity, limiting toxin transport into the blood, which is probably mediated by basolateral Abccs. From an ecotoxicological point of view, elimination of MCLR through this mechanism would reduce the amount of toxin available for trophic transference.

The central objective was to restore the antimicrobial susceptibility in vitro, in strains of Escherichia coli with multidrug resistance phenotype (MDR), from commercial farms by combining antimicrobials with the efflux pump inhibitor 1-(1-naphthylmethyl)-piperazine (NMP). Strains were isolated from the faeces of production and companion animals and from septic tanks. Sensibility profiles were tested to the isolations and 10 MDR strains were obtained. Laboratory strains included two isogenic mutants: AG100A, an RND type pumpdeficient strain and AG112, an acrAB overexpressing strain) and a wild-type strain, AG100. The minimum inhibitory concentrations (MICs) of florfenicol, ciprofloxacin, tetracycline and ampicillin were determined by a serial microdilution method in Luria Bertani (LB) broth in presence or absence of NMP. The highest percentages of resistance were for tetracycline and ampicillin. All combinations of multidrug resistance included tetracycline in their profile. Ampicillin had no effect when combining to NMP. In most of the E. coli MDR strains and in AG112, MICs of ciprofloxacin, florfenicol and tetracycline decreased ≥ 4-fold with NMP. There was no relevant change against wild type strain and MIC remained the same against AG100, independently of NMP´s concentrations. Tetracycline resistance strains strains are probably able to become multirresistant. Ciprofloxacin, tetracycline and florfenicol demonstrated be efflux pumps substrates, but not ampicillin. It is promising the combination of NMP with florfenicol.

Class 1 and 2 integrons are considered the paradigm of multidrug resistant (MDR) integrons. Although class 1 integrons have been found statistically associated to Enterobacteriaceae MDR isolates, this type of study has not been conducted for class 2 integrons. Escherichia coli and 3 species that were found that harbored more than 20% of class 2 integrons in clinical isolates, were selected to determine the role of intI2 as MDR marker. A total of 234 MDR/191 susceptible non-epidemiologically related isolates were analyzed. Seventy-four intI2 genes were found by PCR and sequencing. An intI2 relationship with MDR phenotypes in Acinetobacter baumannii and Enterobacter cloacae was found. No statistical association was identified with MDR E. coli and Helicobacter pylori isolates. In other words, the likelihood of finding intI2 is the same in susceptible and in MDR E. coli and H. pylori strains, suggesting a particular affinity between the mobile element Tn7 and some species. The use of intI2 as MDR marker was species-dependent, with fluctuating epidemiology at geographical and temporal gradients. The use of intI2 as MDR marker is advisable in A. baumannii, a species that can reach high frequencies of this genetic element.

Drug-induced multidrug resistance (MDR) has been linked to overexpression of drug transporting proteins in head and neck squamous cell carcinoma (HNSCC) in vitro. The aim of this work was to reassess these findings in a murine xenograft model. NOD-SCID mice xenotransplanted with 106 HNO97 cells were treated for four consecutive weeks with weekly paclitaxel, biweekly cisplatin (both intraperitoneal), or 5-fluorouracil (5-FU, administered by osmotic pump). Tumor volume and body weight were weekly documented. Expression of drug transporters and Ki-67 marker were examined using quantitative real-time polymerase chain reaction and/or immunohistochemistry. Both paclitaxel and cisplatin significantly reduced tumor volumes after 2–3 weeks. 5-FU-treated animals had significantly lower body weights after 2 or 4 weeks of chemotherapy. None of the drugs affected expression of drug transporters at the mRNA level. However, P-glycoprotein (Pgp) protein expression was increased by paclitaxel (P < 0.01). Ki-67 expression did not change during treatment irrespective of the drug applied. Paclitaxel and cisplatin are effectively tumor volume reducing drugs in a murine xenograft model of HNSCC. Paclitaxel enhanced Pgp expression at the protein level, but not at the mRNA level suggesting transcriptional induction to be of minor relevance. In contrast, posttranscriptional mechanisms or Darwinian selection of intrinsically drug transporter overexpressing MDR cells might lead to iatrogenic chemotherapy resistance in HNSCC.

The human multidrug resistance (MDR) P-glycoprotein (P-gp) mediates the extrusion of chemotherapeutic drugs from cancer cells. Modulators are relevant pharmaceutical targets since they are intended to control or to inhibit its pumping activity. In the present work, a common binding site for Rhodamine 123 and modulators with different modulation activity was found by molecular docking over the crystal structure of the mouse P-gp. The modulators involved a family of compounds, including derivatives of propafenone (3-phenylpropiophenone nucleus) and XR9576 (tariquidar). Our results showed that the relative binding energies estimated by molecular docking were in good correlation with the experimental activities. Preliminary classical molecular dynamics results on selected P-gp/modulator complexes were also performed in order to understand the nature of the prevalent molecular interactions and the possible main molecular features that characterize a modulator. Besides, the results obtained with a human P-gp homology model from the mouse structure are also presented and analyzed. Our observations suggest that the hydrophobicity and molecular flexibility are the main features related to the inhibitory activity. The latter factor would increase the modulator ability to fit the aromatic rings inside the transmembrane domain